Fig 4.
Characterization of proviral clones in vitro. 293T cells (1.5 × 105 cells) were cotransfected with 1 μg wtHTLV-2, HTLV-2ΔAPH, and HTLV-2APHΔBD2 proviral clones or negative-control DNA along with 0.1 μg LTR-2-Luc and 0.01 μg TK-Renilla. All transfections were performed in duplicate and normalized to TK-Renilla to control for the transfection efficiency. Cell lysates or supernatants were harvested 48 h after transfection. Histograms present the average values from three independent experiments. (A) Measurement of Tax activity presented as relative light units (RLU). The asterisk represents the statistically significant difference in Tax activity in HTLV-2ΔAPH and HTLV-2APHΔBD2 proviral clones compared to wtHTLV-2 (P = 0.002 and P = 0.004, respectively, by a Tukey test). (B) Measurement of p19 Gag in the supernatants. (C) Detection of the APH-2 and APH-2 mutant proteins in transfected-cell lysates. The cell lysate was immunoprecipitated by a rabbit anti-APH-2 polyclonal antibody and visualized by WB analysis. β-Actin was assessed as a loading control in those cells transfected with proviral clones only. The asterisks represent a slower-migrating APH detected in both the HTLV-2APHΔBD2 proviral clone and APHΔBD2 cDNA-transfected cells.